Characterizing the Dynamics of Solvated Disaccharides with In-Electrospray Ionization Hydrogen/Deuterium Exchange-Mass Spectrometry.

Carbohydrates have various biological functions that are based on their structures. However, the composition and the glycosidic-bond linkage and configuration of carbohydrates present challenges for their characterization. Furthermore, isomeric features contribute to the formation of intramolecular hydrogen bonds, which influence the flexibility and dynamics of carbohydrates. Hydrogen/deuterium exchange-mass spectrometry (HDX-MS) enables the analysis of protein dynamics by monitoring deuterium labeling after HDX for different lengths of time. In-electrospray ionization (in-ESI) HDX-MS has been used to rapidly label solvated carbohydrates with labeling occurring during desolvation of ESI droplets. Therefore, HDX-labeling times can be altered by changing the spray-solvent conductivity, which changes the initial size of ESI droplets and their resulting lifetimes. Here, we utilize in-ESI HDX-MS to characterize nine isomeric disaccharides with different monosaccharide compositions and glycosidic-bond linkages and configurations. We compared both the relative D-uptake of isomers at individual conductivities, or HDX-labeling times, and the trends associated with labeling at multiple conductivities. Interestingly, the relative D-uptake trends were correlated to isomeric features that affect disaccharide flexibility, including formation of intramolecular hydrogen bonds. Among the isomeric features studied, linkage was observed to have a significant influence on relative D-uptake with (1-3)-linked disaccharides having more change in relative D-uptake with changing conductivity compared to other linkages. Overall, this research illustrates how in-ESI HDX-MS can be applied to structurally characterize disaccharides with distinct isomeric features. Furthermore, this work shows that in-ESI HDX-MS can be used to monitor the dynamics of solvated molecules with rapidly exchanging functional groups.

Effect of pH on In-Electrospray Hydrogen/Deuterium Exchange of Carbohydrates and Peptides.

Carbohydrates are critical for cellular functions as well as an important class of metabolites. Characterizing carbohydrate structures is a difficult analytical challenge due to the presence of isomers. In-electrospray hydrogen/deuterium exchange mass spectrometry (in-ESI HDX-MS) is a method of HDX that samples the solvated structure of carbohydrates during the ESI process and requires little to no instrument modification. Traditionally, solution-phase HDX is utilized with proteins to sample conformational differences, and pH is a critical parameter to monitor and control due to the presence of both acid- and base-catalyzed mechanisms of exchange. For In-ESI HDX, the pH surrounding the analyte changes before and during labeling, which has the potential to affect the rate of labeling for analytes. Herein, we alter the pH of spray solutions containing model carbohydrates and peptides, perform in-ESI HDX-MS, and characterize the deuterium uptake trends. Varying pH results in altered D uptake, though the overall trends differ from the expected bulk-solution trends due to the electrospray process. These findings show the utility of varying pH prior to in-ESI HDX-MS for establishing different extents of HDX as well as distinguishing labile functional groups that are present in different analytes.

Defining the Ceramide Composition of Bovine and Human Milk Gangliosides by Direct Infusion ESI-CID Tandem Mass Spectrometry of Native and Permethylated Molecular Species.

Gangliosides are glycosphingolipids composed of an oligosaccharide that contains one or more sialic acid residues and is linked to a ceramide, a lipid composed of a long chain base (LCB) that bears an amide-linked fatty acyl group (FA). The ceramide portions of gangliosides are embedded in cell membranes; the exposed glycans interact with the extracellular environment. Gangliosides play a myriad of roles in activities such as cell-cell communication, formation of lipid rafts, cellular adhesion, calcium homeostasis, host-pathogen interaction, and viral invasion. Although the epitopes responsible for the interactions of gangliosides are located in the glycan, the epitope presentation is strongly influenced by the orientation of the attached ceramide within the lipid membrane, a feature that depends on the details of its structure, that is, the specific LCB and FA. Since the identities of both the glycan and the ceramide affect the activity of gangliosides, it is important to characterize the individual intact molecular forms. We report here a mass spectrometry-based method that combines the information gained from low-energy collision-induced dissociation (CID) measurements for the determination of the glycan with tandem mass spectra obtained at stepped higher-energy CID for the detailed characterization of the LCB and FA components of intact gangliosides. We provide results from applications of this method to the analysis of gangliosides present in bovine and human milk in order to demonstrate the assignment of LCB and FA for intact gangliosides and differential detection of isomeric ceramide structures.

Formation of Carbohydrate-Metal Adducts from Solvent Mixtures during Electrospray: A Molecular Dynamics and ESI-MS Study.

Electrospray ionization (ESI) is frequently used to produce gas-phase ions for mass spectrometry (MS)-based techniques. The composition of solvents used in ESI-MS is often manipulated to enhance analyte ionization, including for carbohydrates. Moreover, to characterize analyte structures, ESI has been coupled to hydrogen/deuterium exchange, ion mobility, and tandem MS. Therefore, it is important to understand how solvent composition affects the structure of carbohydrates during and after ESI. In this work, we use molecular dynamics to simulate the desolvation of ESI droplets containing a model carbohydrate and observe the formation of carbohydrate adducts with metal ions. Molecular-level details on the effects of formulating mixtures of water, methanol, and acetonitrile to achieve enhanced ionization are presented. We complement our simulations with ESI-MS experiments. We report that when sprayed from aqueous mixtures containing volatile solvents, carbohydrates ionize to form metal-ion adducts rapidly due to rapid solvent evaporation rather than changes in the ionization mechanism. We find that when sprayed from solvent mixtures, carbohydrates are primarily solvated by water due to the migration of more volatile solvents to the surface of the droplet. Ultimately, the structure of the carbohydrate varies depending on its solvent environment, as inter- and intramolecular interactions are affected. We propose that solvents with 25% or more water may be used to enhance the ionization of carbohydrates with minimal effect on the structure during and after ESI.

Distinguishing Carbohydrate Isomers with Rapid Hydrogen/Deuterium Exchange-Mass Spectrometry.

Carbohydrates play key roles in facilitating cellular functions, yet characterizing their structures is analytically challenging due to the presence of epimers, regioisomers, and stereoisomers. In-electrospray-hydrogen/deuterium exchange-mass spectrometry (in-ESI HDX-MS) is a rapid HDX method that samples solvated carbohydrates with minimal instrument modification. When applied to proteins, HDX is often measured after multiple time points to sample the dynamics of structures. Herein, we alter the HDX reaction time by modifying the spray-solvent conductivity, which changes the initial size of ESI droplets, and thus, the droplet lifetimes. We show that this change in droplet lifetime alters the magnitude of HDX for carbohydrate-metal adducts. Furthermore, we illustrate how monitoring HDX at multiple time points enables three trisaccharide isomers (melezitose, maltotriose, and isomaltotriose) to be distinguished. This work illustrates the feasibility of this method for characterizing solvated carbohydrates, including isomeric species which differ only by linkage.

Release of Carbohydrate-Metal Adducts from Electrospray Droplets: Insight into Glycan Ionization by Electrospray.

Glycans have an immense number of biological activities, necessitating increased efforts to characterize glycan structures. Mass spectrometry has been coupled to electrospray ionization (ESI) to characterize carbohydrates. While the gas-phase structures of glycan- and carbohydrate-metal adducts have been characterized, several questions persist concerning the mechanism of transfer of carbohydrates from ESI droplets into the gas phase. Using various computational methods, including molecular dynamics, steered molecular dynamics, and density functional theory calculations, we present a mechanistic investigation on the evaporation of solvent from nanosized droplets, formation of carbohydrate-metal adducts, and their subsequent release into the gas phase. We relate the computational results to mass spectra of melezitose, a model carbohydrate, and its permethylated derivative. Our results confirm two mechanisms for the release of carbohydrate-ion adducts from solvated droplets. Native (unmodified) carbohydrates are ionized via the charged residue model, while the permethylated derivative is ionized via the ion evaporation model. For both mechanisms, the monomer carbohydrate-metal adduct is the dominant species observed. This work illustrates that the ionization mechanisms are dictated by interactions between the carbohydrate and solvent, and coordination of the carbohydrate with the metal ion. Thus, these results provide insight into the molecular interactions that govern the mechanism of release.

Applying an Internal Standard to Improve the Repeatability of In-electrospray H/D Exchange of Carbohydrate-Metal Adducts.

In-electrospray (ESI) hydrogen/deuterium exchange-mass spectrometry (HDX-MS) has been used to characterize solvated carbohydrate structures. However, the rapid exchange rate of hydroxyls, as well as variations in source conditions and ambient humidity, alter the extent of forward and back exchange, resulting in poor repeatability when quantifying D-uptake on different days. Herein, we compare two internal standards, a peptide and derivatized carbohydrate, to improve the repeatability of in-ESI HDX of carbohydrate-metal adducts. Our results show that maltoheptaose, derivatized with Girard's T reagent, is a suitable internal standard for improving the repeatability of in-ESI HDX analyses of carbohydrates of varying size.

Characterization of Electrospray Ionization (ESI) Parameters on In-ESI Hydrogen/Deuterium Exchange of Carbohydrate-Metal Ion Adducts.

The conformations of glycans are crucial for their biological functions. In-electrospray ionization (ESI) hydrogen/deuterium exchange-mass spectrometry (HDX-MS) is a promising technique for studying carbohydrate conformations since rapidly exchanging functional groups, e.g., hydroxyls, can be labeled on the timeframe of ESI. However, regular application of in-ESI HDX to characterize carbohydrates requires further analysis of the in-ESI HDX methodology. For instance, in this method, HDX occurs concurrently to the analyte transitioning from solution to gas-phase ions. Therefore, there is a possibility of sampling both gas-phase and solution-phase conformations of the analyte. Herein, we differentiate in-ESI HDX of metal-adducted carbohydrates from gas-phase HDX and illustrate that this method analyzes solvated species. We also systematically examine the effects of ESI parameters, including spray solvent composition, auxiliary gas flow rate, sheath gas flow rate, sample infusion rate, sample concentration, and spray voltage, and discuss their effects on in-ESI HDX. Further, we model the structural changes of a trisaccharide, melezitose, and its intramolecular and intermolecular hydrogen bonding in solvents with different compositions of methanol and water. These molecular dynamic simulations support our experimental results and illustrate how an individual ESI parameter can alter the conformations we sample by in-ESI HDX. In total, this work illustrates how the fundamental processes of ESI alter the magnitude of HDX for carbohydrates and suggest parameters that should be considered and/or optimized prior to performing experiments with this in-ESI HDX technique. Graphical Abstract ᅟ.

Diverse Reactivity of Dienes with Pentaphenylborole and 1-Phenyl-2,3,4,5-Tetramethylborole Dimer.

The reactions of a monomeric borole and a dimeric borole with 2,3-dimethyl-1,3-butadiene and 1,3-cyclohexadiene were investigated. The monomeric borole reacted at ambient temperature whereas heat was required to crack the dimer to form the monomer and induce reactivity. 2,3-Dimethyl-1,3-butadiene reacts to give diverse products resulting from a cycloaddition process with the B-C moiety of the boroles acting as a dienophile, followed by rearrangements to furnish bicyclic species. For 1,3-cyclohexadiene, a [4+2] process is observed in which 1,3-cyclohexadiene serves as the dienophile and the boroles as the diene partner. The experimental results are corroborated with mechanistic theoretical calculations that indicate boroles can serve as either a diene or dienophile in cycloaddition reactions with dienes.

Mass Spectral Detection of Forward- and Reverse-Hydrogen/Deuterium Exchange Resulting from Residual Solvent Vapors in Electrospray Sources.

Characterizing glycans is analytically challenging since glycans are heterogeneous, branched polymers with different three-dimensional conformations. Hydrogen/deuterium exchange-mass spectrometry (HDX-MS) has been used to analyze native conformations and dynamics of biomolecules by measuring the mass increase of analytes as labile protons are replaced with deuterium following exposure to deuterated solvents. The rate of exchange is dependent on the chemical functional group, the presence of hydrogen bonds, pH, temperature, charge, and solvent accessibility. HDX-MS of carbohydrates is challenging due to the rapid exchange rate of hydroxyls. Here, we describe an observed HDX reaction associated with residual solvent vapors saturating electrospray sources. When undeuterated melezitose was infused after infusing D2O, samples with up to 73% deuterium exchange were detected. This residual solvent HDX was observed for both carbohydrates and peptides in multiple instruments and dependent on sample infusion rate, infusion time, and deuterium content of the solvent. This residual solvent HDX was observed over several minutes of sample analysis and persisted long enough to alter the measured deuterium labeling and possibly change the interpretation of the results. This work illustrates that residual solvent HDX competes with in-solution HDX for rapidly exchanging functional groups. Thus, we propose conditions to minimize this effect, specifically for top-down, in-electrospray ionization, and quench-flow HDX experiments. Graphical Abstract ᅟ.